The blood brain barrier (BBB) separates circulating blood from the cerebrospinal fluid of the brain, and has evolved to protect the brain from any toxins that might be present in the bloodstream. The barrier itself is a three-pronged defensive layer comprising of tight junctions around capillary endothelial cells, a thick basement membrane, and astrocytotic endfeet. These layers only allow the selective diffusion of small hydrophobic molecules such as oxygen, carbon-dioxide, and certain hormones. However, in terms of therapy, the blood brain barrier has always been a hurdle for drugs targeting the brain since these drugs do not cross over in adequate amounts. Recently, a family of peptides called Angiopeps has been shown to be effective in crossing the blood-brain barrier via low-density lipoprotein (LDL) receptor-transport. Moreover, these peptides have been used to transport biomaterials across the BBB via protein-receptor-mediated-transcytosis.

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<center>[[Image:bloodbrainbarrier.png]]</center>

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We aim to tackle BBB entry via DNA origami technology, a relatively novel method of using bottom-up fabrication to create complex nanostructures in a single-step reaction. The entire concept of DNA origami is based on the properties of complementary base-pairing. While top-down fabrication methods have been the sole means of creating nanostructures with high levels of complexity for a while, a recently developed algorithm allowed for the creation of arbitrary two-dimensional nanostructures from a single-strand scaffold of DNA. The basic tenants of this algorithm involve approximating the shape by double-helical DNA strands, interlacing the scaffold to form crossovers, binding oligonucleotide staple strands to fortify the structure, and finally, removing the double. Since the development of caDNAno, a program dedicated solely for the construction of DNA nanostructures, groups have been able to design virtually any 3-D origami structure, including bricks, cages, cylinders, etc.

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<center>[[Image:origami1.png]] [[Image:origami2.png]]</center>

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In their publication, <i>Encapsulation of Gold Nanoparticles in a DNA Origami Cage</i>, Zhao Zhao, Erica Jacovetty, Yan Liu, and Hao Yan demonstrate how to create a rectangular box with a cavity using the bottom-up self-assembly properties of DNA origami. In this particular study, this structure is used to encapsulate gold nanoparticles. This structure allows the surface of the nanoparticles to be tagged with a a controlled number of unique molecules and to control the orientation and intermolecular distance between the nanoparticles. It is stated that a significant advantage in using DNA origami is the spatial resolution of binding sites, on the order of ~6nm, that can be achieved. This structure exhibits a relatively high folding yield and was also found to be fairly robust, withstanding moderate mechanical strain from large nanoparticles placed in the cavity.

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<b>Background</b>

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Zhao Zhao, Erica Jacovetty, Yan Liu, and Hao Yan have demonstrated the ability to create a rectangular box with a cavity using the self assembly properties of DNA origami. This structure exhibits a relatively high folding yield and was also found to be robust enough to survive in a variety of environments. Aside from this particular stricture, DNA origami has a number of inherent advantages over other types of nanoparticles used for similar drug delivery applications. Currently there exists no equivalent technology capable of making a seemingly infinite number of arbitrary shapes like one can with DNA nanotechnology. Another chief advantage is the ease with which the surface of these structures can be functionalized simply by extending oligonucleotides. Functional polymers can be synthesized and then attached to the extended ends. Not only are these structures easy to functionalize, but the number of functional groups that can be attached is limited only by the number of extended oligos, which in our case is on the order of ~100.

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<b>Problem</b>

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With an increasing number of neurological questions still requiring answers, one prominent barrier that remains is a difficulty transporting drugs across the blood-brain-barrier. Creating a vehicle capable of delivering drugs to the brain, concentrating the biodistribution almost entirely to the brain, would be an extremely valuable tool towards advancing treatment for neurological diseases. Additionally, it still is very difficult to study neuronal activity in the brains of monkeys and humans. When working with mice and rats, there exists a wide variety of recording equipment that can be inserted in the brain with ease. IF these particles can be delivered to the brain with ease, they can also be packaged with entities that can record or stimulate neurons.

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<b>Solution</b>

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The aim of our project is to develop a general strategy to functionalize DNA structures with bioactive cues, namely peptides.

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We will demonstrate the utility of this approach with two applications. First, we will attempt to get structures into cells in an organized and controlled fashion, and second, get structures to pass through the blood-brain-barrier and enter the brain through the bloodstream.

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Our approach first involves modifying the ends of an existing box structure with a cavity.

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[[Image:litbox.png]]

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Oligonucleotides are extended from the two ends of the box perpendicular to the plane of the cavity.

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[[Image:2end.png]]

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Once the crude folded structure has been purified to remove stables and misfolded structures, a synthesized carrier polymer can be attached to a complimentary capping sequence that will bind to the extended ends to create functional sites at each extended helix.

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Peptides will then be attached to the carrier polymers and the functionalization process will then be complete.

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<b>Project Goals</b>

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1. Modify the original literature box with extended ends.

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2. Cap the extended ends with a fluorophore to verify functionalization capabilty.

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3. Make the polymer for functionalization and verify binding.

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3. Scale up products.

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4. Test in cells and mice and compare to control.

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Current revision

Boston University

BIOMOD 2013 Design Competition

Introduction

The blood brain barrier (BBB) separates circulating blood from the cerebrospinal fluid of the brain, and has evolved to protect the brain from any toxins that might be present in the bloodstream. The barrier itself is a three-pronged defensive layer comprising of tight junctions around capillary endothelial cells, a thick basement membrane, and astrocytotic endfeet. These layers only allow the selective diffusion of small hydrophobic molecules such as oxygen, carbon-dioxide, and certain hormones. However, in terms of therapy, the blood brain barrier has always been a hurdle for drugs targeting the brain since these drugs do not cross over in adequate amounts. Recently, a family of peptides called Angiopeps has been shown to be effective in crossing the blood-brain barrier via low-density lipoprotein (LDL) receptor-transport. Moreover, these peptides have been used to transport biomaterials across the BBB via protein-receptor-mediated-transcytosis.

We aim to tackle BBB entry via DNA origami technology, a relatively novel method of using bottom-up fabrication to create complex nanostructures in a single-step reaction. The entire concept of DNA origami is based on the properties of complementary base-pairing. While top-down fabrication methods have been the sole means of creating nanostructures with high levels of complexity for a while, a recently developed algorithm allowed for the creation of arbitrary two-dimensional nanostructures from a single-strand scaffold of DNA. The basic tenants of this algorithm involve approximating the shape by double-helical DNA strands, interlacing the scaffold to form crossovers, binding oligonucleotide staple strands to fortify the structure, and finally, removing the double. Since the development of caDNAno, a program dedicated solely for the construction of DNA nanostructures, groups have been able to design virtually any 3-D origami structure, including bricks, cages, cylinders, etc.

In their publication, Encapsulation of Gold Nanoparticles in a DNA Origami Cage, Zhao Zhao, Erica Jacovetty, Yan Liu, and Hao Yan demonstrate how to create a rectangular box with a cavity using the bottom-up self-assembly properties of DNA origami. In this particular study, this structure is used to encapsulate gold nanoparticles. This structure allows the surface of the nanoparticles to be tagged with a a controlled number of unique molecules and to control the orientation and intermolecular distance between the nanoparticles. It is stated that a significant advantage in using DNA origami is the spatial resolution of binding sites, on the order of ~6nm, that can be achieved. This structure exhibits a relatively high folding yield and was also found to be fairly robust, withstanding moderate mechanical strain from large nanoparticles placed in the cavity.